Chimera humanized vascular endothelial growth factor

ABSTRACT

An object of the present invention is to provide a chimera VEGF-E having a reduced antigenicity while maintaining the activity of VEGF-E. The present invention provides a chimera protein having an activity of growing vascular endothelial cells, which is obtained by substituting a part of the sequence of a VEGF analogous protein having an activity of vascularization that binds to KDR (VEGF receptor-2) but does not bind to Flt-1 (VEGF, receptor-1) with a corresponding sequence of a human-derived VEGF analogous protein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of application Ser. No. 10/343,825which is a national stage of PCT/JP01/06856, filed Aug. 9, 2001, whichclaims priority to Japanese Application No. 2000-242629, filed Aug. 10,2000. The disclosures of application Ser. No. 10/343,825 andPC/JP01/06856 are incorporated by reference herein in their entireties.

TECHNICAL FIELD

The present invention relates to chimera vascular endothelial growthfactors (VEGF). More particularly, the present invention relates to achimera protein having an activity of growing vascular endothelialcells, which is obtained by substituting a part of the sequence of aVEGF analogous protein having an activity of vascularization that bindsto KDR (VEGF receptor-2) but does not bind to Flt-1 (VEGF receptor-1)with a corresponding sequence of a human-derived VEGF analogous protein.The present invention also relates to a medicament comprising thechimera protein, DNA encoding the chimera protein, an expression vectorcomprising the DNA, a medicament comprising the expression vector, atransformant having the expression vector, and a process for producingthe chimera protein using the transformant.

BACKGROUND ART

Circulatory disorders caused by vascular system abnormalities areobserved in numerous diseases, and specific examples of such diseasesinclude angina pectoris, heart infarction, lower extremity circulatoryfailure caused by diabetes, and arterial occlusive disease. Thesediseases not only impart a great deal of pain to patients but also oftenlead patients to death. In order to overcome his problem, a great dealof research has been conducted regarding vascularization, and as aresult, vascular endothelial growth factors (hereinafter abbreviated to“VEGF”) and their receptors (Flt-1, KDR) were found to play centralroles therein (Ferrara N. and Davis-Smyth T., Endocrine Review, 18,4-25, 1997, Shibuya A, Advances in Cancer Research, 67, 281-316, 1995).

A possible method to ameliorate various circulatory failures is carriedout by administering VEGF, an analogous protein thereof, or a vector forexpressing them into subcutaneous tissue or muscular tissue of cardiacmuscle or lower extremity to accelerate vascularization by thesevascularization factors. Clinical experiments on VEGF have alreadystated, and VEGF are known to bind to and activate both Flt-1 (VEGFreceptor-1) and KDR (VEGF receptor-2) (Shibuya M. et al. Current Topicsin Microbiology and Immunology, 237, 59-83, 1999). Approximately 90% ofvascularization signals are sent from KDR (VEGF receptor-2) (TakahashiT. et al. Oncogene, 18, 2221-2230, 1999), while Flt-1 (VEGF receptor-1)sends out signals for the chemotaxis of monocytes and macrophages andrelatively weak vascularization signals (Barleon B. et al. Blood, 87,3336-3343, 1996, Clauss M. et al. Journal of Biological Chemistry, 271,17629-17634, 1996). Monocytes and macrophage cells are known to secretevarious cytokines and the like to induce inflammation, and are likely todisturb vascularization therapy. Accordingly, a VEGF analogous proteinwhich binds to only KDR (VEGF receptor-2) and can activate it, isunderstood as more suitable for vascularization therapy. VEGF-E_(NZ-7)was reported as a protein that binds to only KDR (VEGF receptor-2)(Ogawa S. et al. Journal of Biological Chemistry, 273, 31273-31282,1998). Although this gene per se was found in the Orf virus genome ofParapox in 1994, its property still remains unknown, and itscharacteristic features that it binds to only KDR (VEGF receptor-2) andhas potent vascularization activity were elucidated for the first timeby Ogawa et al. (Lyttle D. J. et al. Journal of Virology, 68, 8492,1994; Ogawa S. et al. Journal of Biological Chemistry, 273, 31273-31282,1998).

Thereafter, VEGF-E_(D1701) and VEGF-E_(NZ-2), which are allied withVEGF-E_(NZ-7), were similarly reported as binding to only KDR (VEGFreceptor-2) (FIG. 1) (Meyer M. et al. EMBO Journal 18, 363-374, 1999).As described above, VEGF-E_(NZ-7) binds to only KDR (VEGF receptor-2),has substantially the same vascularization activity as VEGF, and doesnot stimulate the chemotaxis of monocytes and macrophages.

DISCLOSURE OF THE INVASION

As mentioned above, VEGF-E_(NZ-7) is considered to be a protein which isclinically highly applicable, but it has some drawbacks remaining to besolved, i.e., reduction or elimination of antigenicity. Specifically,since the VEGF-E_(NZ-7) gene is not derived from human genome, itsproduct, the VEGF-E_(NZ-7) protein, is regarded as a foreign substancewhich has a certain degree of antigenicity to the human immune system,and thus there is a possibility that antibodies would be generated.Accordingly, although a potent vascularization effect can be expected atthe early stage of administration, if the antibody is generated in theindividual patient it is predicted that VEGF-E_(NZ-7) may be absorbed bythe antibody even though it is readministered and its effect woulddeteriorate.

Specifically, an object of the present invention is to provide a chimeraVEGF-E having a reduced antigenicity while maintaining the activity ofVEGF-E. Further, it is another object of the present invention toprovide a medicament comprising the chimera VEGF-E, DNA encoding thechimera VEGF-E, and a process for producing the chimera VEGF-E using theDNA.

The present inventor has conducted concentrated studies in order toattain the above objects. As a result, they succeeded in reducing andeliminating only antigenicity and foreign property while maintainingVEGF-E_(NZ-7) activity by separating, by means of domain analysis, aportion which is involved in the VEGF-E_(NZ-7) activity from a portionwhich is expected to have relatively high antigenicity, and substitutingthe portion expected to have relatively high antigenicity with a knownhuman protein. This has led to the completion of the present invention.

Thus, the present invention provides a chimera protein having anactivity of growing vascular endothelial cells, which is obtained bysubstituting a part of the sequence of a VEGF analogous protein havingan activity of vascularization that binds to KDR (VEGF receptor-2) butdoes not bind to Flt-1 (VEGF receptor-1) with a corresponding sequenceof a human-derived VEGF analogous protein.

One aspect of the present invention provides a chimera protein having anactivity of growing vascular endothelial cells, which is obtained bysubstituting the amino terminus and/or carboxyl terminus of a VEGFanalogous protein having an activity of vascularization that binds toKDR (VEGF receptor-2) but does not bind to Flt-1 (VEGF receptor-1) withthe amino terminus and/or carboxyl terminus of a human-derived VEGFanalogous protein, respectively. Preferably, there is provided a chimeraprotein obtained by substituting 10 to 50 amino acid residues at theamino terminus of a VEGF analogous protein having an activity ofvascularization that binds to KDR (VEGF receptor-2) but does not bind toFlt-1 (VEGF receptor-1) with the amino terminus of a human derived VEGFanalogous protein and/or substituting 10 to 50 amino acid residues atthe carboxyl terminus of the VEGF analogous protein with the carboxylterminus of the human-derived VEGF analogous protein.

Another aspect of the present invention provides a chimera proteinhaving an activity of growing vascular endothelial cells, which isobtained by substituting a part of the internal sequence of a VEGFanalogous protein having an activity of vascularization that binds toKDR (VEGF receptor-2) but does not bind to Flt-1 (VEGF receptor-1) witha corresponding sequence of a human-derived VEGF analogous protein.Preferably, there is provided a chimera protein which is obtained bysubstituting a part of the sequence of a VEGF analogous protein havingan activity of vascularization that binds to KDR (VEGF receptor-2) butdoes not bind to Flt-1 (VEGF receptor-1) with a human-derived VEGFanalogous protein in such a way that at least the sequences of the loop1 region (amino acid residues 47 to 80) and the loop 3 region (aminoacid residues 89 to 132) of the VEGF analogous protein are maintained.

Preferably, a VEGF analogous protein having an activity ofvascularization that binds to KDR (VEGF receptor-2) but does not bind toFlt-1 (VEGF receptor-1) belongs to the VEGF-E family. More preferably,it is VEGF-E_(NZ-7), VEGF-E_(D1701), or VEGF-E_(NZ-2).

Preferably, a human-derived VEGF analogous protein is PIGF.

Preferably, the chimera protein of the present invention is obtained bysubstituting 10 to 50 amino acid residues at the carboxyl terminus of aVEGF analogous protein having an activity of vascularization that bindsto KDR (VEGF receptor-2) but does not bind to Flt-1 (VEGF receptor-1)with the carboxyl terminus of a human-derived VEGF analogous protein.

Preferably in the chimera protein of the present invention, the VEGFanalogous protein having an activity of vascularization that binds toKDR (VEGF receptor-2) but does not bind to Flt-1 (VEGF receptor-1)belongs to the VEGF-E family, and the loop 1 region of amino acidresidues 47 to 80 and the loop 3 region of amino acid residues 89 to 132in the protein belonging to the VEGF-E family are substantiallyretained.

According to embodiments of the present invention, there is provided achimera protein having any of the following amino acid sequences:

(A) the amino acid sequence as shown in SEQ ID NO: 8, 9, or 10;

(B) amino acid sequence derived from the amino acid sequence as shown inSEQ ID NO: 8, 9, or 10 by deletion, substitution, and/or addition of oneor several amino acids and having an activity of growing vascularendothelial cells; and

(C) an amino acid sequence having 60% or more homology to the amino acidsequence as shown in SEQ ID NO: 8, 9, or 10 and having an activity ofgrowing vascular endothelial cells.

Embodiments of the present invention provide a chimera proteincomprising: any of the amino acid sequences as shown in (A) to (F)below; an amino acid sequence derived from any of the amino acidsequences as shown in (A) to (F) below by deletion, substitution, and/oraddition of one or several amino acids and having an activity of growingvascular endothelial cells; or an amino acid sequence having 60% or morehomology to any of the amino acid sequences as shown in (A) to (F) belowand having an activity of growing vascular endothelial cells.

(A) an amino acid sequence obtained by substituting the sequenceTyr-Leu-Gly-Glu (amino acids 54 to 57 of SEQ ID NO: 7) withAsp-Val-Val-Ser (SEQ ID NO: 21) in the amino acid sequence as shown inSEQ ID NO: 7;

(B) an amino acid sequence obtained by substituting the sequenceSer-Thr-Asn (amino acids 62 to 64) with Glu-Val-Glu in the amino acidsequence as shown in SEQ ID NO: 7;

(C) an amino acid sequence obtained by substituting the sequenceLeu-Gln-Tyr-Asn (amino acids 65 to 68 of SEQ ID NO: 7)) withHis-Met-Phe-Ser (SEQ ID NO: 22) in the amino acid sequence as shown inSEQ ID NO: 7;

(D) an amino acid sequence obtained by substituting the sequenceTrp-Met-Arg-Thr-Leu-Asp-Lys (amino acids 37 to 43 of SEQ ID NO: 7) withPhe-Gln-Glu-Val-Trp-Gly-Arg (SEQ ID NO: 23) in the amino acid sequenceas shown in SEQ ID NO: 7;

(E) an amino acid sequence obtained by substituting the sequenceLys-Pro-Arg-Asp-Thr-Val (amino acids 47 to 52 of SEQ ID NO: 7) withArg-Ala-Leu-Glu-Arg-Leu (SEQ ID NO: 24) in the amino acid sequence asshown in SEQ ID NO: 7; or

(F) an amino acid sequence obtained by substituting the sequenceLeu-Gln-Arg-Ile (amino acids 119 to 122 of SEQ ID NO: 7)) withTyr-Val-Glu-Leu (SEQ ID NO: 25) in the amino acid sequence as shown inSEQ ID NO: 7.

Embodiments of the present invention provide a chimera protein which hasany of the amino acid sequences shown below:

(A) an amino acid sequence as shown in SEQ ID NO: 15;

(B) an amino acid sequence derived from the amino acid sequence as shownin SEQ ID NO: 15 by deletion, substitution, and/or addition of one orseveral amino acids and having an activity of growing vascularendothelial cells, and

(C) an amino acid sequence having 60% or more homology to the amino acidsequence as shown in SEQ ID NO: 15 and having an activity of growingvascular endothelial cells.

Another aspect of the present invention provides a medicament comprisingany of the aforementioned chimera proteins of the present invention.

A further aspect of the present invention provides an activator for KDR(VEGF receptor-2) receptor comprising any of the chimera proteins of thepresent invention.

A further aspect of the present invention provides an agent for growingvascular endothelial cells comprising any of the chimera proteins of thepresent invention.

A further aspect of the present invention provides DNA encoding any ofthe chimera proteins of the present invention.

A further aspect of the present invention provides an expression vectorcomprising the DNA according to the present invention.

A further aspect of the present invention provides a medicamentcomprising the expression vector according to the present invention.

A further aspect of the present invention provides an activator for KDR(VEGF receptor-2) receptor comprising the expression vector according tothe present invention.

A further aspect of the present invention provides an agent for growingvascular endothelial cells comprising the expression vector according tothe present invention.

A further aspect of the present invention provides a transformant havingthe expression vector according to the present invention.

A further aspect of the present invention provides a process forproducing any of the chimera proteins of the present invention whereinthe transformant according to the present invention is used.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows an interrelationship between VEGF-E_(N27) protein and VEGFreceptor.

FIG. 2 shows the structures of VEGF-E_(N27) chimera proteins (VEGF-E-NP,VEGF-F-CP, VEGF-E-NP/CP).

FIG. 3 shows the preparation of VEGF-E_(N27) chimera protein.

FIG. 4 shows the result of the experiment for competitive inhibition ofVEGF-E_(N27) chimera protein on the KDR receptor.

FIG. 5 shows the result of the experiment for assaying theautophosphorylation of KDR receptor.

FIG. 6 shows the result of the experiment on vascular endothelial cellgrowth.

FIG. 7 shows the nucleotide sequence (SEQ ID NO: 16) and the amino acidsequence (SEQ ID NO: 7) of VEGF-E-His.

FIG. 8 shows the nucleotide sequence (SEQ ID NO: 17) and the amino acidsequence (SEQ ID NO: 8) of VEGF-E-NP-His.

FIG. 9 shows the nucleotide sequence (SEQ ID NO: 18) and the amino acidsequence (SEQ ID NO: 9) of VEGF-E-CP-His.

FIG. 10 shows the nucleotide sequence (SEQ ID NO: 19) and the amino acidsequence (SEQ ID NO: 10) of VEGF-E-NP/CP-His.

FIG. 11 shows the nucleotide sequence (SEQ ID NO: 20) and the amino acidsequence (SEQ ID NO: 11) of hPIGF-I-His.

FIG. 12 shows the structures of VEGF-E chimera proteins #19 to #27 and asummary of the results of the experiment for assaying theautophosphorylation of the KDR receptor and the experiment on vascularendothelial cell growth.

FIG. 13 shows the results of the experiment on competitive inhibition onthe KDR receptor using the VEGF-E chimera proteins #19 to #27.

FIG. 14 shows the results of the experiment on vascular endothelial cellgrowth using the VEGF-E chimera proteins #19 to #27.

FIG. 15 shows the structures of VEGF-E chimera proteins #31 to #33 and asummary of the results of the experiment for assaying theautophosphorylation of the KDR receptor and the experiment on vascularendothelial cell growth.

FIG. 16 shows the results of the experiment on competitive inhibition onthe KDR motor using the VEGF-E chimera proteins #31 to #33.

FIG. 17 shows the results of the experiment on vascular endothelial cellgrowth using the VEGF-E chimera proteins #31 to #33.

BEST MODES FOR CARRYING OUT THE INVENTION

Processes and embodiments for carrying out the present invention arehereafter described in detail.

(1) Chimera Protein having an Activity of Growing Vascular EndothelialCells

The chimera protein having an activity of growing vascular endothelialcells according to the present invention can be obtained by substitutinga part of the sequence of a VEGF analogous protein having an activity ofvascularization that binds to KDR (VEGF receptor-2) but does not bind toFlt-1 (VEGF receptor-1) with a corresponding sequence of a human derivedVEGF analogous protein.

The type of VEGF analogous protein having an activity of vascularizationthat binds to KDR (VEGF receptor-2) but does not bind to Flt-1 (VEGFreceptor-1) is not particularly limited, and specific examples thereofinclude a protein belonging to the VEGF-E family. More specific examplesinclude VEGF-E_(NZ-7), VEGF-E_(D1701), and VEGF-E 2 (Ogawa S. et al.Journal of Biological Chemistry, 273, 31273-31282, 1998; and Meyer M. etal. EMBO Journal 18, 363-374, 1999).

In one embodiment of the chimera protein of the present invention, theamino terminus and/or carboxyl terminus of a VEGF analogous proteinhaving an activity of vascularization is substituted with the aminoterminus and/or carboxyl terminus of a human-derived VEGF analogousprotein. The region that is substituted with the human-derived VEGFanalogous protein may be only the amino terminus or only the carboxylterminus of the VEGF analogous protein, or may be both the amino andcarboxyl termini. The length of the amino terminus to be substituted isnot particularly limited as long as the resulting chimera proteinremains the activity of growing vascular endothelial cells. Preferably10 to 50 amino acid residues, more preferably 20 to 40 amino acidresidues, and particularly preferably 20 to 30 amino acid residues atthe amino terminus are substituted with the amino terminus of ahuman-derived VEGF analogous protein. Similarly, the length of thecarboxyl terminus to be substituted is not particularly limited as longas the resulting chimera protein remains the activity of growingvascular endothelial cells. Preferably 10 to 50 amino acid residues,more preferably 10 to 40 amino acid residues, further preferably 10 to30 amino acid residues, and particularly preferably 10 to 20 amino acidresidues at the carboxyl terminus are substituted with the carboxylterminus of a human-derived VEGF analogous protein.

In another embodiment of the chimera protein of the present invention, apart of the internal sequence of a VEGF analogous protein having anactivity of vascularization that binds to KDR (VEGF receptor-2) but doesnot bind to Flt-1 (VEGF receptor-1) is substituted with a correspondingsequence of a human-derived VEGF analogous protein. Preferably, a partof the sequence of a VEGF analogous protein having an activity ofvascularization that binds to KDR (VEGF receptor-2) but does not bind toFlt-1 (VEGF receptor-1) is substituted with a human-derived VEGFanalogous protein in such a way that at least the sequences of the loop1 region (amino acid residues 47 to 80) and the loop 3 region (aminoacid residues 89 to 132) of the VEGF analogous protein are maintained.

In another embodiment of the chimera protein of the present invention,the amino terminus and/or carboxyl terminus of a VEGF analogous proteinhaving an activity of vascularization is substituted with the ammoterminus and/or carboxyl terminus of a human-derived VEGF analogousprotein, and at thy same time, a part of the internal sequence of theVEGF analogous protein is substituted with a corresponding sequence ofthe human-derived VEGF analogous protein.

Preferably, the VEGF analogous protein having an activity ofvascularization according to the present invention belongs to the VEGF-Efamily. VEGF-E is a protein of 149 amino acids, and is composed of:

(I) the amino terminal region comprising a signal peptide (amino acidresidues 1 to 46);

(II) loop 1 region (amino acid residues 47 to 80);

(III) loop 2 region (amino acid residues 81 to 88);

(IV) loop 3 region (amino acid residues 89 to 132); and

(V) the carboxyl terminal region (amino acid residues 133 to 148) (OgawaS. et al., Journal of Biological Chemistry, 273, 31273-31282, 1998).Among the aforementioned regions, since the importance of the loop 3region in binding with KDR (VEGF receptor-2) is suggested, it ispreferred that the chimera protein of the present inventionsubstantially maintains the loop 3 region of amino acid residues 89 to132 of VEGF-E.

The chimera protein of the present invention is obtained by substitutingthe amino terminus and/or carboxyl terminus of a VEGF analogous proteinwith the amino terminus and/or carboxyl terminus of a human-derived VEGFanalogous protein, respectively. This can lower the antigenicity of theVEGF analogous protein. The types of human-derived VEGF analogousproteins are not particularly limited, and examples thereof includePIGF, and particularly, hPIGF1.

The aforementioned chimera proteins may have addition, deletion,substitution, and/or modification in a portion of their amino acidsequence, as long as the proteins remain the activity of growingvascular endothelial cells.

Specific examples of the chimera proteins of the present inventioninclude those having any of the following amino acid sequences:

(A) the amino acrid sequence as shown in SEQ ID NO: 8, 9, 10, or 15 orthe amino acid sequence having the substitution specified herein in theamino acid sequence of SEQ ID NO: 7;

(B) an amino acid sequence derived from the amino acid sequences asdefined in (A) by deletion, substitution, and/or addition of one orseveral amino acids and having an activity of growing vascularendothelial cells; and

(C) an amino acid sequence having 60% or more homology to the amino acidsequences as defined in (A) and having an activity of growing vascularendothelial cells.

The phrase “an amino acid sequence derived from . . . by deletion,substitution, and/or addition of one or several amino acids” used hereinrefers to an amino acid sequence in which, for example, 1 to 20,preferably 1 to 15, more preferably 1 to 10, and further preferably 1 to5 amino acids are deleted, substituted, and/or added.

The term “the amino acid sequence having 60% or more homology to theamino acid sequence” used herein refers to having homology of at least60%. Homology is preferably 70% or more, more preferably 80% or more,further preferably 90% or more, and particularly preferably 95% or more.

The proteins which have an amino acid sequence derived from a certainamino acid sequence by deletion, substitution, and/or addition of one orseveral amino acids and having an activity of growing vascularendothelial cells or an amino acid sequence having 60% or more homologywith a certain amino acid sequence and having an activity of growingvascular endothelial cells, can be produced or obtained using commonrecombinant techniques including site-specific mutagenesis) describedin, for example, Molecular Cloning, A Laboratory Manual, Second Edition,Cold Spring Harbor Laboratory Press (1989), or Current Protocols inMolecular Biology, John Wiley & Sons (1987-1997) and using DNA encodinga protein having an amino acid sequence as shown in any of SEQ ID Nos: 7to 11.

(2) Process for Producing Chimera Protein having an Activity of GrowingVascular Endothelial Cells

A process for producing the chimera protein of the present invention ishereafter described in detail.

A gene encoding a VEGF analogous protein (e.g., the VEGF-E gene) thatbinds to KDR (VEGF receptor-2) but does not bind to Flt-1 (VEGFreceptor-1) and has an activity of vascularization or a modified genethereof is used as a starting material, and DNA fragment is cut outusing a suitable restriction enzyme. Alternatively, a desired DNAfragment which encodes the VEGF analogous protein is prepared by PCR orthe like.

Separately, DNA which encodes the amino terminus and/or carboxylterminus of the human-derived VEGF analogous protein to be substitutedor a partial internal sequence thereof is prepared. The DNA whichencodes the amino terminus and/or carboxyl terminus of the human-derivedVEGF analogous protein or a partial internal sequence thereof may beobtained by cleaving out a desired DNA fragment using a suitablerestriction enzyme from a gene encoding the human-derived VEGF analogousprotein. Alternatively, a desired DNA fragment may by amplified by PCRor may be obtained by chemical synthesis using a DNA synthesizer.

Subsequently, the DNA fragments obtained above can be bound to eachother using a suitable DNA ligase. In this case, an oligonucleotide maybe inserted to align the reading frames, or a part of the nucleotidesequence may be altered to create an identical restriction site.

Any plasmid to which DNA is to be inserted can be used as long as it canbe replicated and retained in a host, and examples include pBR322 andpUC18 derived from Escherichia coli, and pET-3c constructed basedthereon.

Examples of methods for inserting DNA into plasmid include a methoddescribed in T. Maniatis et al., Molecular Cloning, Cold Spring HarborLaboratory, p. 239 (1982).

A cloned gene encoding a chimera protein can be used to construct anexpression vector by ligating the gene downstream of the promoter in avector which is suitable for expression.

Examples of expression vectors that can be used in the present inventioninclude: the plasmid derived from Escherichia coli (pBR322, pBR325,pUC12, pUC13, pET-3); plasmid derived from Bacillus subtilis (pUB110,pTP5, pC194); plasmid derived from yeast (pSH19, pSH15); bacteriophagesuch as λphage or its derivative; animal viruses such as retrovirus andvaccinia virus; and insect viruses (e.g., baculovirus).

The gene may have at its 5′ terminus ATG as a translation initiationcodon, and may have at its 3′ terminus TAA, TGA, or TAG as a translationtermination codon. In order to express the gene, a promoter is connectedupstream thereof. Any promoter can be used in the present invention aslong as it is suitable in respect of the host that is used in the geneexpression.

When the host for transformation is Escherichia coli, trp promoter, lacpromoter, rec A promoter, λPL promoter, Ipp promoter, T7 promoter, andthe like are preferable. When a host is Bacillus subtilis, SP01promoter, SP02 promoter, penP promoter, and the like are preferable.When a host is yeast, PHO5 promoter, PGK promoter, GAP promoter, ADHpromoter, and the like are preferable. When a host is an animal cell,preferable promoters include SV40-derived promoter and a retroviruspromoter.

A vector comprising recombinant DNA having DNA encoding the chimeraprotein of the present invention thus prepared is used to prepare atransformant which has the vector.

Examples of hosts include Escherichia coli (e.g., BL21, BL21(DE3),BL21(DE3)pLysS, and BL21(DE3)pLysE), Bacillus subtilis (e.g., Bacillussubtilis DB105), yeast (e.g., Pichia pastoris, and Saccharomycescerevisiae), animal cells (e.g., COS cell CHO cell, BHK cell, NIH3T3cell, BALB/c3T3 cell, HUVE cell, and LEII cell), and insect cells.

The aforementioned transformation can be carried out in accordance withcommonly employed methods regarding each host. For example, when a hostis Escherichia coli, a vector comprising recombinant DNA is introducedby the heat shock method, electroporation, or the like into a competentcell that was prepared by the calcium method or other methods. When ahost is yeast, a vector comprising recombinant DNA is introduced by theheat shock method, electroporation, or the like into a competent cellthat was prepared by the lithium method or other methods. When a host isan animal cell a vector comprising recombinant DNA is introduced into acell by the calcium phosphate method, lipofection method,electroporation method, or the like.

A transformant which has an expression vector comprising DNA thatencodes the chimera protein of the present invention can be obtained bythe above method. The chimera protein of the present invention can beproduced by culturing the transformant in a suitable medium.

When a transformant is cultured, a commonly used medium can be used foreach host. For example, LB Medium can be used in the case of Escherichiacoli, YPD Medium can be used in the case of yeast, and a medium preparedby adding animal serum to Dulbecco's MEM can be used in the case of theanimal cell.

A transformant can be cultured under general conditions for each host.For example, when a host is Escherichia coli, culturing is conducted atabout 30 to 37° C. for about 3 to 24 hours, and if necessary, aerationor agitation can be applied. When a host is yeast, culturing isconducted at about 25 to 37° C. for about 12 hours to 2 weeks, and ifnecessary, aeration or agitation can be applied. When a host is ananimal cell culturing is conducted at about 32 to 37° C. under 5% CO₂and 100% humidity for about 24 hours to 2 weeks, and if necessary, thecondition of the gas phase can be altered or agitation can be applied.

When the chimera protein of the present invention is expressed using aninsect cell as a host, a protein can be expressed in accordance with themethod described in, for example, Baculovirus Expression Vectors, ALaboratory Manual; Current Protocols in Molecular Biology; andBio/Technology, 6, 47 (1988).

Specifically, a recombinant gene-introduced vector and baculovirus areco-introduced into an insect cell so as to obtain a recombinant virus ina culture supernatant of the insect cell. Thereafter, the insect cell isfurther infected with the recombinant virus, thereby expressing theprotein.

Examples of a vector for introducing the gene that is used in the abovemethod include pVL1392, pVL1393, and pBlueBacIII (all available fromInvitrogen).

As the baculovirus, for example, Autographa californica nuclearpolyhedrosis virus, which is a virus infecting an insect belonging tothe genus Mamestra, can be used.

As the insect cell, for example, ovarian cells of Spodoptera frugiperda,Sf9 and Sf21 (Baculovirus Expression Vectors, A laboratory Manual, W. H.Freeman and Company, New York, 1992) and an ovarian cell of Trichoplusiani, High5, (Invitrogen) can be used.

Examples of methods for co-introducing the recombinant gene-introducedvector and the baculovirus in the insect cell for the preparation of therecombinant virus include the calcium phosphate method (JP PatentPublication (Unexamined Application) No. 2-227075) and lipofectionmethod (Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)).

Medium that can be used for culturing the transformant obtained by usingthe insect cell as a host cell includes commonly used TNM-FH medium(Pharmingen), Sf-900 II SFM medium (GIBCO BRL), ExCell 400 and ExCell405 (JRH Biosciences), and Grace's Insect Medium (Gram, T. C. C.,Nature, 195, 788 (1962)). Culturing is generally conducted underconditions of, for example, pH 6 to 7 at 25 to 30° C. for 1 to 5 days.If necessary, an antibiotic such as gentamicin may be added to themedium during the culture.

In order to extract the chimera protein of the present invention fromthe microorganisms or cell of the aforementioned culture product, thechimera protein may be directly purified from the culture supernatant,or alternatively after culturing of the transformant, microorganisms orcells are disrupted by means of a homogenizer, French press, ultrasound,lysozyme, and/or freeze and thawing to eluate the chimera protein ofinterest extracellularly, and thereby obtain the chimera protein fromthe soluble fraction. When a chimera protein of interest is contained ininsoluble fractions, a method for collecting bacterial bodies or cellscan be performed by first disrupting bacterial bodies or cells,collecting insoluble fractions by centrifugation, and solubilizing themby a buffer comprising guanidine hydrochloride and the like.Alternatively, bacterial bodies or cells are directly disrupted by abuffer comprising a protein denaturing agent such as guanidinhydrochloride to eluate the chimera protein of interest extracellularly.

The chimera protein of the present invention can be purified from theaforementioned soluble fractions by suitably combining conventionalseparation and purification methods. Examples of such methods includesalting-out, solvent precipitation, dialysis, ultrafiltration, gelfiltration, SDS-polyacrylamide gel electrophoresis, ion-exchangechromatography, affinity chromatography, reverse phase high-performanceliquid chromatography, and isoelectric focusing.

When a histidine tag is fused with the chimera protein of the presentinvention and the fused protein is expressed, a nickel carrier such asNi-NTA agarose can be used to specifically adsorb the histidinetag-fused chimera protein for the recovery.

(3) Medicament Comprising a Chimera Protein having an Activity ofGrowing Vascular Endothelial Cells or an Expression Vector ComprisingDNA Encoding a Chimera Protein having an Activity of Growing VascularEndothelial Cells

The chimera protein of the present invention has an activity of growingvascular endothelial cells. Circulatory disorders caused by a vascularsystem abnormality are observed in numerous diseases, and specificexamples of such diseases include angina pectoris, heart infarction,lower extremity circulatory failures caused by diabetes, and arterialocclusive diseases. Administration of the chimera protein of the presentinvention or an expression vector comprising DNA encoding the chimeraprotein to the patients suffering from the aforementioned diseases canimpart therapeutic effects through the growth action on vascularendothelial cells. Since the chimera protein of the present inventioncan activate the KDR (VEGF receptor-2) receptor, it is also useful as atherapeutic or preventive agent for diseases involving abnormality inthe activity of the KDR (VEGF receptor-2) receptor.

As described above, the chimera protein of the present invention isuseful as a medicament.

The chimera protein of the present invention can be formulated intopharmaceutical compositions such as solutions, injections, powders,granules, tablets, suppositories, enteric pills or tablets, and capsulesby using, for example, a pharmaceutically acceptable solvent, excipient,carrier, and adjuvant in accordance with conventional techniques.

The content of the chimera protein of the present invention which is anactive ingredient in the pharmaceutical composition may be about 0000001to 1.0% by weight. These pharmaceutical compositions can be safelyadministered to mammal animals such as human, mouse, rat, rabbit, dog,and cat, and particularly preferably to human as an activator for KDR(VEGF receptor-2) receptor or an agent for growing vascular endothelialcells. Any route of administration such as local, oral, parenteral,intranasal, intravenous, intramuscular, subcutaneous, or percutaneousadministration can be employed.

The dose of the chimera protein of the present invention should beproperly increased or decreased depending on conditions such as age,sex, weight, or symptom of the patient, and route of administration. Forexample, when administered to mammalian animals including human, thechimera protein can be administered in an amount of approximately 0.01μg to 10 mg/kg (body weight) per day.

An expression vector comprising DNA encoding the chimera protein of thepresent invention can be formulated in order to be administered in anyroute of administration such as local, oral, parenteral, intranasal,intravenous, intramuscular, subcutaneous, or percutaneousadministration. Preferably, the expression vector is used in a form ofinjection. For example, the expression vector of the present inventioncan be mixed with a pharmaceutically acceptable excipient to prepare aninjection. Examples of injections include an aseptic solution and anisotonic solution. Alternatively, it can be formulated as a drycomposition, particularly as a freeze-dried composition, and these canbe formulated into an injection by adding sterilized water orphysiological saline at the time of use thereof.

The dose of the expression vector of the present invention should beproperly increased or decreased depending on conditions such as age,sex, weight, or symptom of the patient, and route of administration. Ingeneral, the amount of DNA as an active ingredient is in the range ofabout 1 μg/kg to 1000 mg/kg per day per adult, and preferably in therange of about 10 μg/kg to 100 mg/kg per day per adult.

The present invention will be described in more detail with reference tothe following examples, but the present invention is not limited bythese examples.

EXAMPLES Example 1 Cells and Culture Conditions

Ex-Cell 400 (JRH Biosciences, Lenexa, Kans.) was used as a culturesolution for Sf9 insect cells (Invitrogen, CA, USA). NIH3T3 mousefibroblast and a cell strain NIH3T3-KDR which strongly expresses a humanVEGF receptor, KDR (VEGFR-2), were used in the experiment for assayingthe KDR autophosphorylation by ligand binding. The NIH3T3-KDR cell wasproduced by Sawano et al. (Sawano A. et al. Cell growth andDifferentiation, 7, 213-221, 1996). The NIH3T3 cell and the NIH3T3-KDRcell were maintained in a medium obtained by adding 10% bovine serum, 2mM L-glutamine, and 200 μg/ml G418 (Geneticin; Life Technologies, Inc.,Grand Island, N.Y.) to Dulbecco's Modified Eagle's Medium (DMEM, Nissui,Tokyo). Human umbilical vein endothelial cells (HUVEC, Morinaga, Tokyo)were maintained in HUVE culture medium (Morinaga, Tokyo) and used in theassay for the endothelial cell growth. Recombinant human VEGF₁₆₅ wasforcibly expressed in the Sf9 cells by baculovirus system and thenpurified from the culture supernatant using a haparin column and used.Fc-fused 7N-sKDR (free KDR) was used in the experiment for ligandbinding (Shinkai A. et al. J. Biol. Chem. 273, 31283-31288, 1998).

Example 2 Production of Mutant Chimera VEGF-E_(N27) Proteins VEGF-E-NP,VEGF-E-CP, VEGF-E-NP/CP

The following three types of VEGF-E_(N27)/human PIGF-1 (hereinafterreferred to as “hPIGF-1”) chimera proteins were produced (FIG. 2):

(1) chimera protein VEGF-E-NP prepared by substituting 34 amino acidresidues at the amino terminus of VEGF-E_(N27) with 40 amino acidresidues at the amino terminus of hPIGF-1;

(2) VEGF-E-CP prepared by substituting 18 amino acid residues at thecarboxyl terminus of VEGF-E_(N27) with 21 amino acid residues at thecarboxyl terminus of hPIGF-1; and

(3) VEGF-E-NP/CP prepared by substituting both the amino and carboxylterminuses of VEGF-E_(N27) with those of hPIGF-1.

The above three types of chimera proteins were fused with histidine tagsto simplify the purification. The method in which DNA encoding eachchimera protein was prepared and subcloned into vector pUC18 isdescribed below.

The nucleotide sequence and the amino acid sequence of VEGF-E-His areshown in FIG. 7 and SEQ ID NO: 7, the nucleotide sequence and the aminoacid sequence of VEGF-E-NP-His are shown in FIG. 8 and SEQ ID NO: 8, thenucleotide sequence and the amino acid sequence of VEGF-E-CP-His areshown in FIG. 9 and SEQ ID NO: 9, the nucleotide sequence and the aminoacid sequence of VEGF-E-NP/CP-His are shown in FIG. 10 and SEQ ID NO:10, and the nucleotide sequence and the amino acid sequence ofhPIGF-1-His are shown in FIG. 11 and SEQ ID NO: 11. (VEGF-E-CP andVEGF-E-NP)

pUC18-VEGF-E-His, which is plasmid DNA prepared by subcloning afull-length DNA of the VEGF-E gene fused with a histidine tag at itscarboxyl terminus into pUC18, was cleaved with a restriction enzyme. Afragment (3 kb) that was obtained by cleaving with DraIII and EcoRI anda fragment (about 3 kb) obtained by cleaving with Pf1MI and BamHI werepurified by the gene clean method. Also, 6 oligonucleotides wereproduced and purified by HPLC (Amersham Pharmacia Biotech (Tokyo)):

S155: (SEQ ID NO: 1) 5′-GTGCGAATGCC-3′ S156: (SEQ ID NO: 2)5′-CATTCGCACTTT-3′ S150: (SEQ ID NO: 3)5′-GGCCTCTGCGGGAGAAGATGAAGCCGGAAAGGTGCGGCGATGCTGTTCCCCGGAGGCACCATCACCATCACCATTAAG-3′ S151: (SEQ ID NO: 4)5′-AATTCTTAATGGTGATGGTGGATGGTGCCTCCGGGGAACAGCATCGCCGCACCTTTCCGGCTTCATCTTCTCCCGCAGAGGCCGG-3′ S166: (SEQ ID NO: 5)5′-GATCCATGCCGGTCATGAGGCTGTTCCCTTGCTTCCTGCAGCTCCTGGCCGGGCTGGCGCTGCCTGCTGTGCCCCCCCAGCAGTGGGCCTTGTCTGCTGGGAACGGCTCGTCAGAGGTGGAAGTGAATGA-3′ S167: (SEQ ID NO: 6)5′-TTCACTTCCACCTCTGACGAGCCGTTCCCAGCAGACAAGGCCCACTGCTGGGGGGGCACACGAGGCAGCGCCAGCCCGGCCAGGAGCTGCAGGAAGCAAGGGAACAGCCTCATGACCGGCATG-3′

S150 and S151, S155 and S156, and S166 and S167 (10 μg/ml each) wereboiled under conditions of 10 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 1 mMDTT, and 100 mM NaCl for 10 minutes, and then allowed to stand at roomtemperature for annealing. Subsequently, a DNA fragment comprisingVEGF-E purified by the gene clean method and the annealedoligonucleotides were ligated to each other by DNA ligase, followed bymolecular cloning on the plasmid vector. The DNA fragment ofDraIII/EcoRI and oligonucleotides of S150/S151 and S155/S156 wereligated for VEGF-E-CP by DNA ligase. The DNA fragment of Pf1MI/BamHI andoligonucleotides of S166/S167 were ligated for VEGF-E-NP by DNA ligase.Thus, a histidine tag was fused to the carboxyl terminus. Thefull-length DNAs which encoded VEGF-E-CP and VEGF-E-NP were subclonedinto the pUC18 plasmid vector. The plasmids were respectively designatedas pUCE-CP and pUCE-NP.

(VEGF-E-NP/CP)

pUCE-CP and pUCE-NP were Cleaved with BsrGI and EcoRI and 200 bp and 3kb DNA fragments respectively obtained therefrom were purified by thegene clean method, followed by ligation with DNA ligase. Thus, ahistidine tag was fused to the carboxyl terminus. The full-length DNAwhich encoded the VEGF-E-NP/CP was subcloned into pUC18, and thisplasmid DNA was designated as pUCE-NP/CP.

Subsequently, each of the subcloned DNAs was cleaved out and subclonedinto a baculovirus vector. Each DNA of pUCE-CP, pUCE-NP and pUCE-NP/CPwas cleaved with BamHI and EcoRI and 450 bp fragments were purified bythe gene clean method. The fragments were cloned into the BamHI andEcoRI site in the multicloning sites of pVL1393 (Invitrogen) which is abaculovirus vector. These plasmid DNAs were designated as pVL3E-CP,pVL3E-NP, and pVL3E-NP/CP.

Example 3 Preparation of VEGF-E Chimera Protein

By using DNAs of pVL3E-CP, pVL3E-NP and pVL3E-NP/CP and Baculogold(Pharmingen, San Diego, Calif.) of baculovirus DNA, transfection intoSf9 cells was performed by lipofectin met Recombinant viruses having DNAencoding the chimera protein were amplified by repeating incubation for3 to 4 days. In order to express the chimera protein, infection with therecombinant virus Sf9 cells was carried out at the multiplicity ofinfection (MOI) (i.e., the number of virus particles per cell) of 10.After the incubation for 3 days, a culture solution to which the chimeraprotein had been secreted was collected.

Subsequently, the culture solution was concentrated. In order toconcentrate 150 ml of culture solution to 15 ml, an agitation-typeultrafilter device (MILLIPORE, USA) was used. The concentrate wassubjected to dialysis in 20 mM sodium phosphate buffer (pH 8.0), 10 mMimidazole, 300 mM NaCl, and 20% glycerol in order to negatively chargethe histidine tag that was fused with the recombinant protein.

After the dialysis, the histidine tag-fused protein was specificallyadsorbed using Ni-NTA agarose (QIAGEN, Germany). The Ni-NTA agarose towhich the recombinant protein had been adsorbed was introduced into acolumn and washed with 20 mM sodium phosphate buffer (pH 8.0), 50 mMimidazole, 300 mM NaCl, and 20% glycerol, and the chimera protein ofinterest was eluted using 20 mM sodium phosphate buffer (pH 8.0), 250 mMimidazole, 300 mM NaCl, and 20% glycerol. Finally, the eluate wassubjected to dialysis in 20 mM sodium phosphate buffer (pH 7.4), 50 mMNaCl, and 20% glycerol. The purified chimera protein was electrophoresedin 15% SDS-PAGE and analyzed by Coomassie staining and Western blottingusing anti-histidine tag monoclonal antibody (anti-His-tagmAb),anti-VEGF-E serum, and anti-PIGF antibody. The purification level of thechimera protein was 80% or higher (FIG. 3).

Example 4 Experiment on Competitive Inhibition of VEGF-E Chimera Proteinon KDR Receptor

Fc-fused 7N-sKDR was diluted to 3 μg/ml in physiological saline(hereinafter abbreviated to “PBS”), 50 μl thereof was added to each wellof a 96-well cell culture plate (Immunon (registered trademark) 2HB,DYNEX TECHNOLOGIES, INC, VA), allowed to stand at 4° C. for 12 hours,and _(s)KDR was then adsorbed on the plate. The plate was washed twicewith a blocking buffer (PBS containing 1% bovine serum albumin), andblocking was carried out using the blocking buffer at 25° C. for 30minutes. In the experiment on competitive inhibition, 50 μl of samplecontaining 16 μg/ml of ¹²⁵I-radioactively labeled VEGF (1,200-1,800Ci/mmol; Amersham Pharmacia Biotech) and 16-1,000 μg/ml of chimeraprotein was added to the _(s)KDR-coated wells, a reaction was carriedout at 25° C. for 3 hours, and the wells were then washed with ablocking buffer. The ¹²⁵I-radioactively labeled VEGF bound to _(s)KDR ofeach well was assayed using a γ-counter.

The results showed that the three types of chimera VEGF-E_(N27) proteins(VEGF-E-NP, VEGF-E-CP, VEGF-E-NP/CP) efficiently inhibited the bindingof VEGF to KDR. This strongly indicates that the chimera VEGF-E_(N27)protein binds to KDR with high affinity (FIG. 4).

Example 5 Experiment for Assaying Autophosphorylation of KDR Receptor

In order to confirm that the chimera VEGF-E_(N27) proteins directly bindto and activate KDR, an experiment was carried out to assay theautophosphorylation of the KDR receptor by these proteins. Before theNIH3T3-KDR cells were stimulated with the chimera proteins, the cellswere cultured until 60% confluent. Thereafter, the cells were exposed tolow-concentration serum conditions in DMEM containing 0.5% bovine serumovernight. The cells were stimulated by each of the chimera proteins ata concentration of 1 to 500 ng/ml at 37° C. for 5 minutes. The cellswere washed twice in ice-cooled PBS containing 0.1 mM Na₃VO₄, and thendissolved with a 1% Triton X-100 buffer solution (50 mM HEPES pH 7.4,150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl, 2% trasylol, 1mM PMSF, 50 mM NaF, 10 mM Na₄P₂O₇, 2 mM Na₃VO₄). The dissolved samplewas centrifuged at 15,000 rpm for 10 minutes, and the supernatant wascollected. The protein concentration in the supernatant was assayedusing the Bio-Rad Protein Assay Kit (Richmond, Calif.), and anequivalent protein was used in the analysis. In the immunoblotting foranalyzing the autophosphorylated KDR, the dissolved sample waselectrophoresed in 7.5% SDS-PAGE and then transferred to anitrocellulose membrane. The transferred nitrocellulose membrane wasblocked with a blocking buffer (a washing buffer containing 5% BSA [20mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20]), and binding reactionwas then carried out with a blocking buffer containing a primaryantibody.

Signals obtained by Western blotting were analyzed using horseradishperoxidase-conjugated secondary antibodies and enhancedchemiluminescence reagents (ECL, Amersham).

As a result, the three types of chimera VEGF-E_(N27) proteins(VEGF-E-NP, VEGF-E-CP, VEGF-E-NP/CP) were found to activate the tyrosinekinase of the KDR receptor in a dosage-dependent manner (FIG. 5).

Example 6 Experiment for Vascular Endothelial Cell Growth

The HUVEC (Morinaga, Tokyo) which were cultured in the medium for HUVEculture (Nissui) containing growth factors, FGF, added thereto, wereinoculated on a 24-well plate (CellTite C-1 Plate 24F, Akita SumitomoBakelite Co., Ltd., Akita) in amounts of 4,000 cells, and allowed tostand in an incubator at 37° C. for 4 hours to adsorb the cells. Thechimera VEGF-ED protein was directly added to the medium to a finalconcentration of 50 ng/ml, and cultured for 3 days. The cells were fixedwith formalin and then stained with Crystal Violet. The number of cellswas counted, and relatively compared to analyze the endothelial cellgrowth. The results showed that the chimera protein had the equivalentactivity of endothelial cell growth as the VEGF-E protein in vitro (FIG.6).

(Summary of Examples 1 to 6)

From the above results, the three types of chimera VEGF-E_(N27) proteins(VEGF-E-NP, VEGF-E-CP, VEGF-E-NP/CP) were found to bind to KDR with highaffinity, accelerate the autophosphorylation of KDR, and stimulate thevascular endothelial cell growth. The antigenicity of the protein isknown to be relatively strong at free termini such as an ammo orcarboxyl terminus, however, both termini of VEGF-E were found to beunnecessary for activating KDR tyrosine kinase and could be substitutedwith a human-derived protein such as PIGF used in the ExampleAccordingly, these chimera VEGF-E_(N27) proteins (VEGF-E-NP, VEGF-E-CP,VEGF-E-NP/CP) are considered to be usable as safer vascularizationfactors than the original VEGF-E_(N27) proteins.

Example 7 Production of Chimera VEGF-E Proteins #19 to #27 and #31 to#33

(1) Production of Chimera VEGF-E Proteins #19 to #27

The amino acid sequence of the substituted region is shown below. Thenucleotide sequence of PIGF was used for that of the substituted region.Structures of the chimera VEGF-E proteins #19 to #27 are shown in FIG.12.

#19: the amino acid sequence from residues #54-57 (YLGE) of VEGF-E (SEQID NO: 7) is substituted with the amino acid sequence (DVVS) (SEQ ID NO:21) of the corresponding region in human PIGF.#20: the amino acid sequence from residues #62-64 (STN) of VEGF-E (SEQID NO: 7) is substituted with the amino acid sequence (EVE) of thecorresponding region in human PIGF.#21: the amino acid sequence from residues #65-68 (LQYN) of VEGF-E (SEQID NO: 7) is substituted with the amino acid sequence (HMFS) (SEQ ID NO:22) of the corresponding region in human PIGF.#22: the amino acid sequence from residues #3743 (WMRTLDK) of VEGF-E(SEQ ID NO: 7) is substituted with the amino acid sequence (FQEVWGR)(SEQ ID NO: 23) of the corresponding region in human PIGF.#23: the amino acid sequence from residues #47-52 (KPRDTV) (β chain #1)of VEGF-E (SEQ ID NO: 7) is substituted with the amino acid sequence(RALERL) (SEQ ID NO: 24) of the corresponding region in human PIGF.#24: the amino acid sequence from residues #98-101 (VTVS) (β chain #6,between cysteine 6 and cysteine 7) of VEGF-E (SEQ ID NO: 7) issubstituted with the amino acid sequence (MQLL) (SEQ ID NO: 26) of thecorresponding region in human PIGF.#25: the amino acid sequence from residues #102-104 (VTG) (a portion ofloop 3) of VEGF-E (SEQ ID NO: 7) is substituted with the amino acidsequence (KTR) of the corresponding region in human PIGF.#26: the amino acid sequence from residues #119-122 (LQRI) (β chain #7,11 amino acid residues upstream region from the 7th cysteine) of VEGF-E(SEQ ID NO: 7) is substituted with the amino acid sequence (YVEL) (SEQID NO: 25) of the corresponding region in human PIGF.#27: the amino acid sequence from residues #111-118 (TNSGVSTN)(VEGF-E-specific amino acid sequence) of VEGF-E (SEQ ID NO: 7) isremoved (deletion mutation).

(2) Production of Chimera VEGF-E Proteins #31 to #33

The structures of the chimera proteins are shown below as nucleotidesequences. The structures of chimera VEGF-E proteins #31 to #33 areshown in FIG. 15.

Boxed portions: cysteine sites (the 2nd, 3rd, 4th, and 5th)

Underlined portions: VEGF-E sequences

Fundamental nucleotide sequence: PIGF-I

#31 (SEQ ID NO: 12): the amino acid sequence between the 2nd cysteineand the 3rd cysteine of human PIGF is substituted with the correspondingamino acid sequence of VEGF-E

#32 (SEQ ID NO: 13): the amino acid sequence between the 4th cysteineand the 5th cysteine of human PIGF is substituted with the correspondingamino acid sequence of VEGF-E

#33 (SEQ ID NO: 14): the amino acid sequence between the 2nd cysteineand the 3th cysteine and the amino acid sequence between the 4thcysteine and the 5th cysteine of human PIGF are substituted with thecorresponding amino acid sequences of VEGF-E

DNA fragments having the above sequences were constructed byconventional gene recombinant techniques, and VEGF-E chimera proteins(#19 to #27 and #31 to #33) were prepared in the same manner as inExample 3.

Example 8 Physiological Activities of Chimera VEGF-E Proteins #19 to #27and #31 to #33

The VEGF-E chimera proteins (#19 to #27 and #31 to #33) prepared inExample 7 were used to perform an experiment on competitive inhibitionon the KDR receptor (in the same manner as in Example 4), an experimentfor assaying autophosphorylation of the KDR receptor (in the same manneras in Example 5), and an experiment on vascular endothelial cell growth(in the same manner as in Example 6).

FIG. 12 shows the structures of VEGF-E chimera proteins #19 to #27 andsummarized results of the experiment for assaying theautophosphorylation of the KDR receptor and the experiment on vascularendothelial cell growth. FIG. 13 shows the assay result obtained by theexperiment on competitive inhibition on KDR receptor. FIG. 14 shows theassay result obtained by the experiment on vascular endothelial cellgrowth.

FIG. 15 shows the structures of VEGF-E chimera proteins #31 to #33 and asummary of the results of the experiment for assaying theautophosphorylation of KDR receptor and the experiment of vascularendothelial cell growth. FIG. 16 shows the assay result obtained by theexperiment on competitive inhibition on KDR receptor FIG. 17 shows theassay result obtained by the experiment on vascular endothelial cellgrowth.

As is apparent from the results shown in FIGS. 12 to 14, it was foundthat all the chimera proteins other than #24, #25, and #27 bind to KDRwith high affinity, accelerate the autophosphorylation of KDR, andstimulate the growth of vascular endothelial cells. Accordingly, it wasclarified that not only both termini of VEGF-E but also some internalsequences in VEGF-E can be substituted with human-derived protein suchas PIGF.

As is apparent from the results shown in FIGS. 15 to 17, it was foundthat #33, which was obtained by substituting the amino acid sequencebetween the 2nd cysteine and the 3rd cysteine in human PIGF thatexhibits no autophosphorylation of KDR receptor and no vascularendothelial cell growth (including loop 1 in the core region of humanPIGF) and the amino acid sequence between the 4th cysteine and the 5thcysteine (including loop 3 in the core region of human PIGF) with thecorresponding amino acid sequences of VEGF-E (respectively includingloop 1 and loop 3 in the core region of VEGF-E), accelerates theautophosphorylation of KDR and stimulates the growth of vascularendothelial cells. Accordingly, loop 1 and loop 3 in the core regionpossibly play a role in the expression of physiological activities ofVEGF-E.

INDUSTRIAL APPLICABILITY

The chimera VEGF-E of the present invention can be used as a safevascularization factor since its antigenicity is reduced whilemaintaining the activity of growing vascular endothelial cells.

1. A chimera protein which has any of the amino acid sequences shownbelow: (A) the amino acid sequence which is obtained by deleting 1-20amino acids of signal peptide at the amino-terminal region of the aminoacid sequence as shown in SEQ ID NO: 15, or (B) the amino acid sequenceas shown in (A), in which one to ten amino acids are deleted,substituted, and/or added and which has an activity of growing vascularendothelial cells.
 2. A chimera protein which has any of the amino acidsequences shown below: (A) the amino acid sequence which is obtained bydeleting 6 amino acids of histidine-tag at the carboxyl-terminal regionfrom the amino acid sequence as shown in SEQ ID NO: 15, or (B) the aminoacid sequence as shown in (A), in which one to ten amino acids aredeleted, substituted, and/or added and which has an activity of growingvascular endothelial cells.
 3. A chimera protein which has any of theamino acid sequences shown below: (A) the amino acid sequence which isobtained by deleting 1-20 amino acids of signal peptide at theamino-terminal region and 6 amino acids of histidine-tag at thecarboxyl-terminal region from the amino acid sequence as shown in SEQ IDNO: 15, or (B) the amino acid sequence as shown in (A), in which one toten amino acids are deleted, substituted, and/or added and which has anactivity of growing vascular endothelial cells.
 4. The chimera proteinaccording to claim 3, wherein the amino acid sequence is obtained bydeleting 1-15 amino acids of signal peptide at the amino-terminal regionand 6 amino acids of histidine-tag at the carboxyl-terminal region fromthe amino acid sequence as shown in SEQ ID NO:
 15. 5. The chimeraprotein according to claim 3, wherein the amino acid sequence isobtained by deleting 1-10 amino acids of signal peptide at theamino-terminal region and 6 amino acids of histidine-tag at thecarboxyl-terminal region from the amino acid sequence as shown in SEQ IDNO:
 15. 6. The chimera protein according to claim 3, wherein the aminoacid sequence is obtained by deleting 1-5 amino acids of signal peptideat the amino-terminal region and 6 amino acids of histidine-tag at thecarboxyl-terminal region from the amino acid sequence as shown in SEQ IDNO: 15.